Abstract Core C Antibodies (Abs) are essential and ubiquitous tools for biologic research, including for identifying the temporal and spatial pattern of expression in cells and human or animal tissues, identifying interacting partners and their biologic activities, facilitating Xray or EM structure determination and for exploring the role of proteins in disease in pre-clinical models. The goal of Core C is to identify and provide recombinant monoclonal antibodies (rAbs) that bind to the multiple conformations of ATP Binding Cassette (ABC) transporter family members and to enable all four Projects in the P01 that use antibodies to functionally characterize these transporters, determine their various structures and model them as they progress through the transport cycle. We will generate multiple recombinant antibody fragments (Fabs) for use in single molecule fluorescent imaging (Project 1), as fiducial markers in single particle cryo-EM studies (Project 2), and as crystallization partners to accelerate crystallization (Project 3). Finally, data from antibody epitopes, structural data involving antibodies from the Projects and the pharmacologic effect of antibody binding will be inputs for molecular modeling (Project 4) and will provide significant insights into the mechanism of transporter function. The transporters undergo conformational changes but direct structural evidence for the entire transport cycle is lacking. We have developed Fab phage display libraries and methods for selection and validation of Fabs to conformational changes of enzymes and transporters. Based on our success in generating conformationally- specific Fabs, we expect similar success in identifying Fabs for additional states of TmrAB and for the other transporters, namely TAPL, MRP4 and when available, TAP1/2. Conformationally-specific Abs that bind ABC transporters are not available commercially; thus, new recombinant antibodies are needed to fully characterize the intermediate structures in the pumping cycle. The Craik lab has a record of generating recombinant antibodies to a variety of proteins including ABC transporters and has demonstrated the utility of recombinant antibody fragments (Fabs) as fiducial markers for single particle cryoEM studies of protein. Given the availability of Fabs to TmrA/B and other ABC transporters, similar cryoEM studies will be performed to determine structures of different conformations. High-resolution structures could result from rigid Fab- membrane protein complexes. Our aims are to 1) Generate recombinant Fabs and scFv or nanobodies to transporters in multiple conformational states by panning using existing Fab, scFv or nanobody libraries. 2) Express and purify sufficient quantities of Fabs for characterization and for structural and functional studies. Since producing appropriate antigens is one of the most difficult challenges for creating Fabs that recognize conformational epitopes, Core C will work closely with the Core B and Project 1 with shared personnel to apply our library panning methodology to different states of ABC transporters.